Summary Complete genome duplication requires a close collaboration between the replication machinery and many regulatory factors. These factors can assist replisomes in coping with large numbers of template obstacles, and their defects are frequently associated with human diseases such as cancer and DNA damage syndromes. It is our long-term goal to understand how replication regulatory proteins interact with the replisome and aid replication. We and others have shown that the evolutionarily conserved Smc5/6 complex is essential for promoting replication under normal and DNA damaging conditions. During the last two cycles of this grant, we have made significant progress in elucidating the structure of this complex, its unique SUMO E3 activity, and its multiple effects on genome maintenance in the budding yeast model system. Importantly and most relevant to the current proposal, we recently showed that Smc5/6 and its binding partner, the scaffold protein Rtt107, comprise a new pathway that aids large replicon synthesis. Large replicons are difficult to duplicate and strongly associated with fragile sites and other forms of genomic instability. Investigating the mechanisms by which Smc5/6 and Rtt107 facilitate large replicon synthesis will broaden our understanding of the maintenance of high-risk genomic regions. We found that Smc5/6 and Rtt107 interact with DNA polymerase and helicase complexes and contribute to their sumoylation. Moreover, these sumoylation events influence replication. Based on these findings, we hypothesize that Smc5/6 and Rtt107 directly promote replisome function to aid large replicon synthesis. Extending from this work, we showed that Rtt107 is the hub of a protein network composed of Smc5/6 and other genome maintenance factors. How Rtt107 supports the interactions within this network and how each interaction contributes to replication are important questions to address. In the next funding cycle, we propose to test our hypothesis stated above by examining how Smc5/6 and Rtt107 affect replication fork functions during large replicon synthesis, and define the biochemical features of the Smc5/6 and Rtt107 interactions with DNA polymerase and helicase complexes. In addition, we will determine how the sumoylation of DNA polymerase and helicase complexes affects the initiation and elongation stages of replication. Findings from these studies will shed light on the roles of Smc5/6, Rtt107, and SUMO in promoting the completion of large replicon synthesis. Finally, we will investigate the biochemical basis of Rtt107-mediated interactions and the functions of each interaction. To achieve these goals, we will use a combination of genetic, biochemical, biophysical approaches in the highly effective yeast system. Outcomes of the proposed work will expand our view on how large replicon synthesis is achieved, how SUMO regulates replication, and how the Rtt107 interactome contributes to genome duplication. As such, these studies will have broad implications in our understanding of genome maintenance mechanisms.